The present invention relates to alpha-7 nicotinic acetylcholine receptor (α7 nAChR) chimeric receptors containing one or more regions homologous to a nicotinic cholinergic receptor and a serotoninergic receptor for measuring α7 nAChR function and methods and compositions useful in the identification of α7 nAChR agonists, antagonists and allosteric modulators.
Ion channels are hydrophilic pores across the cellular membrane that open in response to stimulation to allow specific inorganic ions of appropriate size and charge to pass across the membrane. Depending on the nature of the ligand, ion channels expressed in the plasma membrane are broadly classified as voltage-gated ion channels (VGIC) or ligand-gated ion channels (LGIC) where the ligand usually is considered to be an extracellular messenger such as a neurotransmitter (Gopalakrishnan and Briggs, 2006). Specific residues in ion channel proteins also determine the specificity for the inorganic ion transported including sodium, potassium, calcium, and chloride ions.
Ligand-gated ion channels are essential in mediating communication between cells. These channels convert a chemical signal (often a neurotransmitter, as for example, acetylcholine) released by one cell into an electrical signal that propagates along a target cell membrane through specific ion influx. A variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. Numerous families of ligand-gated receptors have been identified and categorized by their specific ligands and on the basis of sequence identity. These include receptors specific for acetylcholine, glutamate, glycine, GABA A, and 5-HT.
nAChRs receptors, members of the cys-loop superfamily of LGIC, are widely characterized transmembrane proteins involved in the physiological responses to the neurotransmitter ACh and are distributed throughout both the central nervous system (CNS) and the peripheral nervous system (PNS). The nicotinic acetylcholine receptors (nAChRs) are multiunit proteins of neuromuscular and neuronal origins and mediate synaptic transmission between nerve and muscle and between neurons upon interaction with the neurotransmitter acetylcholine (ACh). Organizationally, nAChRs are homopentamers or heteropentamers composed of nine alpha and four beta subunits that co-assemble to form multiple subtypes of receptors that have a distinctive pharmacology. ACh is the endogenous ligand (agonist), while nicotine is a prototypical agonist that non-selectively activates all nAChRs. Functional nAChRs are widely expressed in the central nervous system and in the ganglia of the autonomic nervous system. nAChRs are involved in a range of synaptic and extra synaptic functions. In the peripheral nervous system, nAChRs mediate ganglionic neurotransmission whereas in the CNS, nicotinic cholinergic innervation mediates fast synaptic transmission and regulates processes such as transmitter release, synaptic plasticity and neuronal network integration by providing modulatory input to a range of other neurotransmitter systems. Thus, nAChR subtypes are implicated in a range of physiological and pathophysiological functions related to cognitive functions, learning and memory, reward, motor control, arousal and analgesia.
The α7 nAChR is a ligand-gated calcium channel formed by a homopentamer of α7 subunits. These receptors are expressed in several brain regions, especially localized at presynaptic and postsynaptic terminals in the hippocampus and cerebral cortex, regions critical to the synaptic plasticity underlying learning and memory. Presynaptic α7 nAChRs present on GABAergic, glutamatergic and cholinergic neurons can facilitate directly or indirectly the release of neurotransmitters such as glutamate, GABA and norepinephrine whereas postsynaptic receptors can modulate other neuronal inputs and trigger a variety of downstream signaling pathways. This facilitation of pre- and post-synaptic mechanisms by α7 nAChRs could influence synaptic plasticity, important for cognitive functions involved in attention, learning, and memory. Support for this hypothesis has emerged from preclinical studies with selective agonists, antagonists, and more recently, positive allosteric modulators (PAMs). Structurally diverse α7 nAChR agonists such as PNU-282987, SSR-180711A and AR-R17779 can improve performance in social recognition (Van Kampen, M. et. al., 2004), maze training (Levin, E. D. et. al., 1999; Arendash, G. W. et. al, 1995) and active avoidance (Arendash, G. W. et. al, 1995) models while α7 nAChR antagonists or antisense impair such performance (Bettany, J. H. et. al., 2001; Felix, R. and Levin, E. D., 1997; Curzon, P. et. al., 2006). Both agonists and PAMs, exemplified respectively by PNU-282987 and PNU-120596, have also been shown to reverse auditory gating deficits in animal models (Hajos, M. et. al., 2005; Hurst et al, 2005).
Although α7 nAChRs have significant Ca2+ permeability comparable to NMDA receptors, these receptors do not require membrane depolarization for activation, and the current responses are curtailed by rapid receptor desensitization processes (Quick, M. W., and Lester, R. A. J., 2002). The functional significance of α7 nAChRs is not only attributable to its electrogenic properties (i.e. modulation of neuronal excitability and neurotransmitter release) but also to its high Ca2+-permeability and association with biochemical signaling pathways. Thus, activation of α7 nAChR can result in increased intracellular Ca2+, leading to signal transduction cascades involving the activation of a variety of protein kinases and other proteins by phosphorylation. Proteins that are phosphorylated in response to α7 nAChR activation could include extracellular signal-regulated kinase ½ (ERK1/2) (Ren, K. et. al., 2005), cAMP response element binding protein (CREB) (Roman, J. et. al., 2004) and Akt (Shaw, S. H. et. al., 2002).
The rapid receptor desensitization (within 50-100 milliseconds) of α7 nAChRs greatly limits the development of functional assays required for measurement of channel activity. A simple and high throughput assay is critical for screening for ligands that interact with the α7 nAChR with potential for the treatment of diseases where cognitive deficits remain an underlying component.
Serotonin (5-hydroxytryptamine, or 5-HT) receptors belong to at least two superfamilies: G-protein-associated receptors and ligand-gated ion channels. The majority of 5-HT receptors couple to effector molecules through G-protein coupled receptors. However, the 5-HT3 receptor functions as a rapidly activating ion channel and, like other LGIC family members, incorporates a nonselective cation channel in its primary structure. 5-HT3 receptors are expressed in native central and peripheral neurons where they are thought to play important roles in sensory processing and control of autonomic reflexes (Richardson, B. P., et al., 1985). 5-HT3 channels desensitize much slower than α7 nAChR.
Therefore, a chimeric receptor prepared from the human N-terminal ligand binding domain of α7 nAChR and the pore forming C-terminal domain of the human 5-HT3 would preserve the ligand selectivity for human α7 nAChR while delay the desensitization of the receptor. The delayed desensitization would make it easier to measure the channel function of α7 nAChR. Other amino acid stretches containing different segments of the α7 nAChR could be introduced to generate additional chimeras. Such chimeric receptors would be particularly useful for functional screening and identifying novel α7 nAChR agonists, modulators and antagonists.